For integral lysosomal membrane proteins, specific peptide sequences are essential for their delivery to the lysosomes. These are the M6P-independent targeting systems that rely on peptide sequences and not M6P or other carbohydrate recognition systems. Inherent in the original concept of the lysosome, as described by deDuve (1), is an essential mechanism to localize or target specific proteins/enzymes to this subcellular organelle. Goldstein and Brown were awarded a Nobel Prize for their discovery and elucidation of specific receptors on cells for the uptake of extracellular low-density lipoproteins into the cell and delivery to the lysosomes (2, 3). Later this concept of receptor-mediated endocytosis was generalized to include not only extracellular ligand-receptor interactions, but also the essential need for such a system within cells for delivery/targeting of newly synthesized proteins to the lysosomes. One such system was elucidated by Kornfeld and coworkers (4) and Sly and coworkers (5, 6) and is now known as the mannose-6-phosphate (M6P) system. This M6P is used by many of the soluble proteins for their delivery to the lysosomes. The deficiency of the ability to attach M6P to such proteins results in the secretion of several dozen proteins from the cell and a progressive multisystem fatal disease termed MLII/MLIII (7).
M6P and mannose receptor systems form the essential basis for the delivery of administered recombinant proteins for the treatment of the so-called lysosomal storage diseases (8). Production of lysosomal proteins for delivery to specific affected tissues is completely dependent upon understanding what receptor is critical to those tissues for uptake, internalization, and delivery of the therapeutic proteins to the lysosomes (8). For example, the mannose-receptor is significantly represented on macrophages and other cells of the reticuloendothelial system (RES). Gaucher disease has its major disease manifestations in macrophages and the RES. Effective enzyme therapy was developed based on the ability to create recombinant acid β-glucosidase, the defective enzyme in Gaucher disease, with mannose terminated oligosaccharide residues (9, 10). For Fabry disease, Pompe disease and Mucopolysaccharidoses I, II, and VI, recombinant enzymes with M6P oligosaccharides have become the standards of care for the treatment of these diseases (e.g. 11, 12). The successes in treating these formerly fatal and severely debilitating diseases have been documented extensively. The total sales of enzymes for the treatment of these diseases now exceed $5 B annually.
There remain several membrane associated, not integral membrane, proteins that appear to have a completely different system for targeting to the lysosomes. One such protein is an enzyme, termed acid β-glucosidase, which is defective in Gaucher disease, the most common lysosomal storage disease (13). Acid β-glucosidase (also referred to as D-glucosyl-N-acylsphingosine glucohydrolase, glucocerebrosidase, GCase, assigned Enzyme Commission No. EC 3.2.1.45) is a lysosomal exoglycosidase for β-glucose-terminated sphingolipids (14,15). Insufficient activity of GCase is causal to the variants of Gaucher disease, a common lysosomal storage disease (16). The human or mouse genes, GBA1 or Gba1, respectively, are about 7.5 kb and contain 11 exons, which encodes a highly (˜86% identical/92% similar) conserved amino acid sequence. Over 300 mutations of various types have been found in association with the variants of Gaucher disease and some have clear prognostic value for affected patients (17). Each of the resultant different single amino acid substitutions lead to GCases with altered catalytic, stability, or both defects (e.g., 18). GCase is translated from mRNAs into a protein that contains two functional, in tandem, leader sequences that differ in length, either 39 (SEQ ID NO 24) or 19 amino acids (19). The preferred initiation codon is not known.
Mature GCase is a glycoprotein of 497 amino acids (FIG. 3, SEQ ID NO 2) that is produced by co-translational glycosylation of four of five N-glycosylation sequences (N463 not occupied) of which only N-19 is essential for the formation of a catalytically active conformer (20). The enzyme also has properties of a membrane associated, but not transmembrane, protein. Unlike most soluble lysosomal proteins that are trafficked to the lysosome by the mannose-6-phosphate (M6P) receptor system (21,22), GCase contains little if any M6P (23). Newly synthesized unglycosylated GCase is not secreted out of cells nor is enzyme secreted from I-Cell fibroblasts, which are deficient in the enzyme needed for M6P (21). Thus, the targeting to the lysosome of newly synthesized, intracellular, GCase is not oligosaccharide dependent (24).
Like the M6P targeting systems, non-carbohydrate-mediated lysosomal targeting disruptions lead to multisystem fatal diseases (8, 9). Lysosomal Integral Membrane Protein 2 (LIMP-2) has been identified as a trafficking receptor for GCase (23, 25, 26). LIMP-2 is a 478 amino acid, 85 Kd glycoprotein protein that is also known as SCARB-2/CD36L2. This protein is present in the ER, Golgi, endosomal, lysosomal, and plasma membrane compartments of cells (26, 27). As the name indicates, LIMP-2 is an integral membrane protein with N- and COOH-terminal transmembrane domains, and a luminal domain (ldLIMP-2) that binds GCase (23) and potentially other proteins. LIMP-2 binds to GCase in the ER (pH-6.8) and the enzyme remains bound to LIMP-2 during its transport through the Golgi, trans-Golgi network, and endosomal compartments. LIMP-2 delivers GCase to the lysosomes after an acidic pH-modulated dissociation of the receptor and ligand in the late endosomal compartment with liberation of GCase. This dissociation is mediated by LIMP-2 histidine 171 (28). No other proteins have been identified to bind to LIMP-2 inside of cells.
LIMP-2/SCARB-2 is also a scavenger receptor on the plasma membrane that binds peptide sequences of viruses, in particular enteroviruses (e.g. EV71), for internalization, lysosomal delivery and degradation (29-32). The ligand amino acid sequence of EV71 for human LIMP-2 has been identified within VP1 (30), which has no homology to GCase sequences. The corresponding receptor sequence on LIMP-2 is between amino acids 144-151 (28). Other LIMP-2/SCARB-2 protein ligands that bind at the plasma membrane include KCNQ1, KCNE2, and Megalin (33).
Humans and mice with mutations in the LIMP-2 gene develop characteristic neurologic and renal diseases, but do not exhibit gross findings of Gaucher disease, i.e., GC storage or Gaucher cells (33, 34). The human diseases associated with LIMP-2 mutations are termed the action myoclonus-renal failure syndrome (34). LIMP-2 deficient cells in humans and mice exhibit excess secretion of GCase out of the cells and into plasma or culture media, but little GC accumulation in tissues (34, 23). LIMP-2 variants have also been implicated as potential modifiers in the development of Parkinson/Alzheimer diseases (35, 36, 33), as have GBA1 mutations (36-39). Disruption of appropriate trafficking of GCase to the lysosome may provide a mechanistic basis for the impact of GBA1/Gba1 mutations in the modification of α-synuclein metabolism and its role in Parkinson disease (37, 38, 40). The impact of LIMP-2 on the expression of Gaucher disease and both GCase and LIMP-2 variants as modifiers of synucleinopathies highlight the importance of understanding the interactions of GCase and LIMP-2 and the localization of synthesized GCase to the lysosome.
Thus, there remains a need for methods and compositions effective for treating lysosomal storage diseases related to defects in the acid β-glucosidase pathway such as Gaucher disease. Further, there is a need for treatments of disease states related to dysregulation of synuclein metabolism that may result from, or be exacerbated by, disruption of appropriate trafficking of GCase to the lysosome, such as neurodegenerative disease states including Parkinson disease, Alzheimer disease, and Lewy body disease. Finally, there remains a need for improved methods of synthesizing recombinant GCase, which can subsequently be used to treat disease states resulting from deficiencies in this enzyme. The instant disclosure seeks to address one or more of these needs.